CD4+ T cell-derived IL-22 enhances liver metastasis by promoting angiogenesis.

Metastasis is a cancer-related systemic disease and is responsible for the greatest mortality rate among cancer patients. Interestingly, the interaction between the immune system and cancer cells seems to play a key role in metastasis formation in the target organ. However, this complex network is only partially understood. We previously found that IL-22 produced by tissue resident iNKT17 cells promotes cancer cell extravasation, the early step of metastasis. Based on these data, we aimed here to decipher the role of IL-22 in the last step of metastasis formation. We found that IL-22 levels were increased in established metastatic sites in both human and mouse. We also found that Th22 cells were the key source of IL-22 in established metastasis sites, and that deletion of IL-22 in CD4+ T cells was protective in liver metastasis formation. Accordingly, the administration of a murine IL-22 neutralizing antibody in the establishment of metastasis formation significantly reduced the metastatic burden in a mouse model. Mechanistically, IL-22-producing Th22 cells promoted angiogenesis in established metastasis sites. In conclusion, our findings highlight that IL-22 is equally as important in contributing to metastasis formation at late metastatic stages, and thus, identify it as a novel therapeutic target in established metastasis.

When less is more: Experimental Bishop-Koop technique for reduction in the use of laboratory animals for intestinal pathophysiological studies.

The use of animals to gain knowledge and understanding of diseases needs to be reduced and refined. In the field of intestinal research, because of the complexity of the gut immune system, living models testing is mandatory. Based on the 3Rs (replacement, reduction and refinement) principles, we aimed to developed and apply the derived-intestinal surgical procedure described by Bishop and Koop (BK) in rats to refine experimental gastrointestinal procedures and reduce the number of animals used for research employing two models of intestinal inflammation: intestinal ischemia-reperfusion injury and chemical-induced colitis. Our results show the feasibility of the application of the BK technique in rodents, with good success after surgical procedure in both small and large intestine (100% survival, clinical recovery and weight regain). A considerable reduction in the use of the number of rats in both intestinal inflammation models (80% in case of intestinal ischemia-reperfusion damage and 66.6% in chemical-induced colitis in our experimental design) was achieved. Compared with conventional experimental models described by various research groups, we report excellent reproducibility of intestinal damage and functionality, survival rate and clinical status of the animals when BK is applied.

Experimental study to assess the impact of vasopressors administered during maintenance of the brain-dead donation in the quality of the intestinal graft.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3+ counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3+ mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE (p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver (p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3 + counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3 + mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE ( p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver ( p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

Cupremia y colesterolemia como factores de riesgo en pacientes con enfermedad de Alzheimer / Plasma levels of copper and cholesterol as risk factors in Alzheimer disease patients / Cupremia e colesterolemia como fatores de risco em pacientes com doença de Alzheimer

Se estudiaron 241 personas, 119 controles y 122 pacientes con enfermedad de Alzheimer (EA) subagrupados en tres categorías de acuerdo con el estadio clínico de la dolencia, con el objetivo de investigar la influencia de niveles elevados de cobre libre y colesterol plasmático como factores de riesgo para la EA. Las conclusiones obtenidas de los resultados indicaron que los pacientes expuestos a una combinación de alto colesterol y de cobre no unido a ceruloplasmina tuvieron mayor proporción de marcadores de estrés oxidativo (carbonilos proteicos, sustancias reactivas al tiobarbiturato, glutatión oxidado y descenso de antioxidantes totales en sangre), conjuntamente con un incremento de HDL-colesterol peroxidado y lipoproteína "a" que correlacionó con la gravedad de su cuadro. Lo mismo sucedió con la relación entre péptidos amiloides (1-40) y (1-42) en plasma y los valores del mini-test de estado cognitivo (MMSE). Se halló que una función de adición de efectos que cuantificó el daño por cobre libre y colesterol oxidado resultó directamente proporcional a la pérdida de desempeño cognitivo estimada por medio del MMSE. Esta función es de fácil determinación y puede considerarse un nuevo biomarcador para estudiar riesgo en poblaciones expuestas, apoyar el diagnóstico clínico, o evaluar la eficacia de estrategias terapéuticas en pacientes con EA.

Alzheimer disease (AD) patients (122) compared to control subjects (119) were studied to determine the role of chronic exposure of hypercholesterolemic plasma levels and free copper (not bound to ceruloplasmin) as biomarkers of progression for AD. Oxidative stress parameters, lipid profile, amyloid levels, and cognitive status were studied in all participants. Conclusions obtained indicated that patients exposed simultaneously to free copper and increased cholesterol levels present higher indicators of oxidative stress (protein carbonyls, thiobarbituric acid-reactive substances, decreased total antioxidant activity in plasma and elevated oxidized HDL-cholesterol). Lipoprotein "a" concentrations also correlated with the clinical progression of the disease. The ratio amyloid ß(1-40)/ß(1-42) in plasma inversely correlated with the cognitive performance estimated by the Mini-Mental State Examination Test (MMSE). A defined function that weighs the contribution of the deleterious effect produced by combined free copper and Ox-HDL-cholesterol exposure directly correlated with the loss of cognitive performance. Thus, this biomarker could be a new tool for the screening of large populations under risk, or may be a useful way to estimate the efficacy of therapeuti approaches in patients suffering AD.

Foram estudadas 241 pessoas, 119 controles e 122 pacientes com doença de Alzheimer (DA), agrupados em três categorias de acordo com o estágio clínico da doença, com o objetivo de investigar a influência de níveis elevados de cobre livre e colesterol plasmático como fatores de risco para a DA. As conclusões obtidas a partir dos resultados indicaram que os pacientes expostos a uma combinação de colesterol alto e de cobre não ligados à ceruloplasmina apresentaram maior proporção de marcadores de estresse oxidativo (carbonilos proteicos, substâncias reativas ao tiobarbiturato, glutationa oxidada e diminuição dos antioxidantes totais no sangue ), juntamente com um aumento da HDL-colesterol peroxidado e lipoproteína "a" que correlacionou com a gravidade de sua condição. O mesmo aconteceu com a relação entre os peptídeos amilóides (1-40) e (1-42) em plasma e os valores do mini-teste do estado cognitivo (MMSE). Verificou-se que uma função de adição de efeitos que quantificou o dano por cobre livre e colesterol oxidado resultou diretamente proporcional à perda de desempenho cognitivo estimada através do MMSE. Esta função é fácil de determinar e pode ser considerada um novo biomarcador para estudar o risco em populações expostas, apoiar o diagnóstico clínico ou avaliar a eficácia de estratégias terapêuticas em pacientes com DA.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model.

BACKGROUND: Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function. METHODS: ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. RESULTS: The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. CONCLUSIONS: Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

Donor preconditioning with rabbit anti-rat thymocyte immunoglobulin ameliorates ischemia reperfusion injury in rat kidney transplantation.

A major concern in transplantation is the preservation of organ function. Ischemia time and microcirculatory disturbance of the organ cannot be avoided and may result in ischemia reperfusion injury (IRI), increasing the risk of delayed graft function (DGF) and acute and chronic rejection. Anti-thymocyte immunoglobulin (rATG) is a polyclonal antibody preparation with multiple effects when administered to recipients. Our objective has been to evaluate whether the administration of rATG to kidney donors instead of recipients, in an experimental model of syngeneic rat transplantation, ameliorates IRI and facilitates immediate graft function recovery. Urea and creatinine levels and necrosis severity scores were significantly lower in kidneys from donors that had received rATG (urea: control: 211±8mg/dl vs. treatment: 110±15mg/dl, p<0.001; creatinine: control: 4.6±0.24mg/dl vs. treatment: 2.6±0.22mg/dl, p<0.001; necrosis severity scores: control: 2.3 vs. treatment: 1.6, p<0.05). TUNEL staining showed 80±13 positive cells in control group and 9±3 (p<0.001) in treatment group. In situ expression of proinflammatory cytokines TNF-α, IL-6, IL-21 and TGF-ß1 was reduced in rATG group (p<0.01); the same was observed for KIM-1 and caspase 8 (p<0.001). Cytoprotective genes Bcl2 and HO-1 were upregulated in situ in treatment group (p<0.001). In situ expression of IL-17, caspase 9, IL-23a, CxCl3 and ICAM1 showed no difference between groups (p>0.05). Findings suggest ATG administered to donors may ameliorate the IRI process in kidney transplantation, expressed by lower necrosis and apoptosis scores and the improvement of renal function, which may be explained through the diminished in situ expression of inflammatory mediators.

Inmunosupresión en donantes renales: Expresión de citoquinas proinflamatorias / Immunosuppression in kidney donors with rapamycin and tacrolimus: Proinflammatory cytokine expression

La lesión por isquemia y reperfusión (IRI) es uno de los principales problemas en el trasplante. Nuestro objetivo fue evaluar el efecto del pre - acondicionamiento al donante con rapamicina y tacrolimus para prevenir la lesión por IRI. Las ratas Wistar donantes, 12 horas antes de la nefrectomía, recibieron fármacos inmunosupresores. La muestra se dividió en cuatro grupos experimentales: un grupo con intervención simulada (sham), un grupo control sin tratamiento, otro tratado con rapamicina (2 mg/kg) y el restante tratado con tacrolimus (0.3 mg/kg). Se retiró el riñón izquierdo y después de tres horas de isquemia fría, se lo trasplantó. Veinticuatro horas después, el órgano trasplantado se recuperó para el análisis histológico y la evaluación de la expresión de citoquinas. El tratamiento de pre-acondicionamiento con rapamicina o con tacrolimus redujo significativamente el nitrógeno ureico en sangre y los niveles de creatinina en comparación con el control (BUN: p < 0.001; creatinina: p < 0.001). La necrosis tubular aguda fue significativamente menor en las ratas donantes tratadas con inmunosupresores en comparación con el grupo control (p < 0.001). Finalmente, las citoquinas inflamatorias, como TNF-α, IL-6 y rIL-21, mostraron niveles más bajos en el injerto de los animales que recibieron tratamiento. Este estudio experimental exploratorio muestra que el pre-acondicionamiento en donantes con rapamicina y tacrolimus en dos grupos distintos mejora los resultados clínicos y anatomopatológicos en receptores, con una reducción in situ de citoquinas pro-inflamatorias relacionadas con la diferenciación Th17, y de este modo crea un ambiente favorable para la diferenciación de células T regulatorias (Tregs).

The ischemia-reperfusion injury (IRI) remains a major problem in transplantation. The objective of this study was to evaluate the effects of preconditioning a donor group with rapamycin and another donor group with tacrolimus to prevent IRI. Twelve hours before nephrectomy, donor Wistar rats received immunosuppressive drugs. The sample was divided into four experimental groups: a sham group, an untreated control group, a group treated with rapamycin (2 mg/kg) and a group treated with tacrolimus (0.3 mg/kg). Left kidneys were removed and, after three hours of cold ischemia, grafts were transplanted. Twenty-four hours later, the transplanted organs were recovered for histological analysis and evaluation of cytokine expression. The pre-conditioning treatment with rapamycin or tacrolimus significantly reduced donor blood urea nitrogen and creatinine levels compared with control group (BUN: p < 0.001 vs. control and creatinine: p < 0.001 vs. control). Acute tubular necrosis was significantly lower in donors treated with immunosuppressant drugs compared with the control group (p < 0.001). Finally, inflammatory cytokines such as TNF-α, IL-6 and rIL-21 showed lower levels in the graft of pre-treated animals. This exploratory experimental study shows that preconditioning donors with rapamycin and tacrolimus in different groups improves clinical outcome and pathology in recipients and reduces in situ pro-inflammatory cytokines associated with Th17 differentiation, creating a favorable environment for the differentiation of regulatory T cells (Tregs).

[Immunosuppression in kidney donors with rapamycin and tacrolimus. Proinflammatory cytokine expression]. / Inmunosupresión en donantes renales. Expresión de citoquinas proinflamatorias.

The ischemia-reperfusion injury (IRI) remains a major problem in transplantation. The objective of this study was to evaluate the effects of preconditioning a donor group with rapamycin and another donor group with tacrolimus to prevent IRI. Twelve hours before nephrectomy, donor Wistar rats received immunosuppressive drugs. The sample was divided into four experimental groups: a sham group, an untreated control group, a group treated with rapamycin (2 mg/kg) and a group treated with tacrolimus (0.3 mg/kg). Left kidneys were removed and, after three hours of cold ischemia, grafts were transplanted. Twenty-four hours later, the transplanted organs were recovered for histological analysis and evaluation of cytokine expression. The pre-conditioning treatment with rapamycin or tacrolimus significantly reduced donor blood urea nitrogen and creatinine levels compared with control group (BUN: p < 0.001 vs. control and creatinine: p < 0.001 vs. control). Acute tubular necrosis was significantly lower in donors treated with immunosuppressant drugs compared with the control group (p < 0.001). Finally, inflammatory cytokines such as TNF-a, IL-6 and rIL-21 showed lower levels in the graft of pre-treated animals. This exploratory experimental study shows that preconditioning donors with rapamycin and tacrolimus in different groups improves clinical outcome and pathology in recipients and reduces in situ pro-inflammatory cytokines associated with Th17 differentiation, creating a favorable environment for the differentiation of regulatory T cells (Tregs).

Protective effect of immunosuppressive treatment before orthotopic kidney autotransplantation.

BACKGROUND: Ischemia reperfusion injury (IRI) is one of the risk factors for delayed graft function, acute rejection and long term allograft survival after kidney transplantation. IRI is an independent antigen inflammatory process that produces tissue damage. Our objective was to study the impact of immunosuppressive treatment (IS) on IRI applying only one dose of IS before orthotopic kidney autotransplantation. METHODS: Twenty-four rats allocated in four groups were studied. One group served as control (G1: autotransplanted rats without IS) and the rest received IS 12 h before kidney autotransplantation (G2: Rapamycin, G3: Mycophenolate mofetil and G4: Tacrolimus). RESULTS: Improved renal function and systemic inflammatory response were found among IS groups compared to the control group (Delta Urea p<0.0001; Delta Creatinine p<0.0001; Delta C3 p<0.001). The number of apoptotic nuclei in renal medulla in G1 was higher than in IS groups (p<0.0001). Tubular damage was less severe in IS groups respecting G1 (p<0.001). C3, TNF-α and IL-6 expression in kidney samples was reduced when IS was used compared to the control group. No differences were observed among the different immunosuppressive drugs tested. However, Heme oxygenase-1(HO-1) was increased only in Rapamycin treatment. CONCLUSIONS: These data suggest that the use of IS administered before transplant attenuates the IRI process after kidney transplantation in an animal model.

Down-regulation of intestinal epithelial innate response by probiotic yeasts isolated from kefir.

Kefir is obtained by milk fermentation with a complex microbial population included in a matrix of polysaccharide and proteins. Several health-promoting activities has been attributed to kefir consumption. The aim of this study was to select microorganisms from kefir able to down-regulate intestinal epithelial innate response and further characterize this activity. Caco-2 cells stably transfected with a human CCL20 promoter luciferase reporter were used to screen a collection of 24 yeast and 23 bacterial strains isolated from kefir. The Toll-like receptor 5 agonist, flagellin was used to activate the reporter cells, while pre-incubation with the selected strains was tested to identify strains with the capacity to inhibit cell activation. In this system, 21 yeast strains from the genera Saccharomyces, Kluyveromyces and Issatchenkia inhibited almost 100% of the flagellin-dependent activation, whereas only some lactobacilli strains showed a partial effect. K. marxianus CIDCA 8154 was selected for further characterization. Inhibitory activity was confirmed at transcriptional level on Caco-2/TC-7 and HT-29 cells upon flagellin stimulation. A similar effect was observed using other pro-inflammatory stimulation such as IL-1beta and TNF-alpha. Pre-incubation with yeasts induced a down-regulation of NF-kappaB signalling in epithelial cells in vitro, as well as expression of other pro-inflammatory chemokines such as CXCL8 and CXCL2. Furthermore, modulation of CCL20 mRNA expression upon flagellin stimulation was evidenced in vivo, in a mouse ligated intestinal loop model. Results indicate kefir contains microorganisms able to abolish the intestinal epithelial inflammatory response that could explain some of the properties attributed to this fermented milk.

Efectos de la eliminación de la tensión mediante puntos epineurales: estudio comparativo empleando diferentes técnicas de sutura en un modelo animal / Effects of tension elimination by using epineural anchoring stitches: a comparative study employing different techniques of suture in an experimental model

Objetivo. Documentar la utilidad de una técnica de sutura epineural bajo tensión, en comparación con la sutura mediante injerto interpuesto y la neurorrafia termino-terminal, en un estudio realizado en un modelo experimental animal (rata Wistar). Material y Método. Un lote de 20 animales, considerados como 40 unidades fisiológicas independientes a nivel del miembro inferior, fueron separados en 4 grupos de 10: en el primero se realizó una sutura bajo tensión con puntos epineurales a nivel del nervio ciático luego de resecar 4 mm de nervio. En el segundo se realizó una sutura término-terminal sin tensión, en el tercero se colocó un puente de injerto autólogo de 4 mm de extensión y el cuarto fue utilizado como control. A los 90 días de efectuados los procedimientos, se reexpusieron las zonas operadas y se documentaron las respuestas fisiológicas a los diferentes tipos de sutura mediante potenciales de acción de músculo (PAM) y nervio (PAN). Resultados. El promedio de la velocidad de conducción para la sutura simple fue el mejor de los tres grupos operados (14,60 mm/ ms), aunque peor que el control (19,31 mm/ms). La sutura a tensión mediante puntos epineurales (12,02 mm/ms) demostró resultados neurofisiológicos superiores que cuando se utilizó injerto autólogo (11,09 mm/ms). Conclusión. El presente estudio demuestra que si se logra coaptar un nervio ciático de rata mediante sutura epineural con puntos distales a la línea de neurorrafia, sus resultados son mejores que al emplear injerto. Sin embargo, estos resultados, obtenidos en un modelo de animal pequeño, deben ser cuidadosamente extrapolados a la práctica quirúrgica habitual.

Objective. To measure the utility of this technique, in comparison with interposed grafts and termino-terminal neuroraphy, all applied in an experimental model (Wistar rat).Materials and method. 20 rats were used in both sides (40 legs, each one considered as independent physiological units, were grouped in four groups: in the first one, a section of thesciatic nerve was performed, a segment 4mm long was discarded and an epineural suture with distal anchoring stitches was done under slight tension. In the second group a tensionlesstermino-terminal neuroraphy was performed after sciatic nerve section. In the third group a 4 mm long graft was employed and the fourth was used as control. 90 days after this procedure, the animals were reoperated and muscle and nerve action potentialswere recorded in both sides. Results. The mean conduction velocity for the simple suture was higher than the other two groups, but lower than the control(19,31 mm/ms). Neuroraphy with epineural stitches (12,02 mm/ms) was better than when an autologous nerve graft was employed (11,09 mm/ms).Conclusion. This study demonstrates that a good result can be obtained if a nerve is sutured under slight tension, employing epineural anchoring stitches. Nevertheless, more studies shouldbe performed before applying these results to human nerve suture.

Erythrocyte membrane, plasma and atherosclerotic plaque lipid pattern in coronary heart disease.

The objective was to analyze the lipid composition of the atherosclerotic plaque (AP), plasma and erythrocyte membrane (EM) in patients with advanced coronary heart disease (CHD). AP were obtained through endarterectomy in 18 patients. Ten normolipemic healthy subjects were selected to obtain the normal lipid pattern profile. Total lipids of AP and EM were determined by HPTLC, and the fatty acid profile from AP, EM and plasma using TLC-FID. The relative amount of the lipid species analyzed in AP was in line with the data in the literature [phospholipids: 23.5 mol% +/- 3.5; total cholesterol 68.9 mol% +/- 7.9; triglyceride 7.6 mol% +/- 3.4]. Plasma and EM from CHD patients compared to controls, showed a decrease in polyunsaturated fatty acids and an increase in saturated fatty acids leading to a decrease in the unsaturation index (plasma: 1.67 +/- 0.06 vs. 1.28 +/- 0.03, P < 0.05; EM: 2.28 +/- 0.04 vs. 1.25 +/- 0.010, P < 0.05) and an enhancement in the saturated/unsaturated ratio (plasma: 0.35 +/- 0.02 vs. 0.52 +/- 0.02, P < 0.05; EM: 0.45 +/- 0.01 vs. 0.83 +/- 0.04, P < 0.05). These data are consistent with an essential fatty acid deficiency. Total cholesterol was increased in the CHD's EM (32.3 +/- 0.8 vs. 40.6 +/- 2.5, P < 0.05) with a decrease in phospholipid percentage (67.7 +/- 0.7 vs. 59.4 +/- 2.6, P < 0.05) indicating an alteration in membrane fluidity. These findings suggest changes in EM lipids in CHD patients in spite of different pathological conditions such as age, smoking status and diabetes. The analysis of the lipid composition of EM could provide a useful tool to monitor the evolution of the CHD.

Erythrocyte membrane, plasma and atherosclerotic plaque lipid pattern in coronary heart disease / Perfil lipídico de membrana de eritrocito, plasma y placa ateromatosa en la enfermedad coronaria

The objective was to analyze the lipid composition of the atherosclerotic plaque (AP), plasma and erythrocyte membrane (EM) in patients with advanced coronary heart disease (CHD). AP were obtained through endarterectomy in 18 patients. Ten normolipemic healthy subjects were selected to obtain the normal lipid pattern profile. Total lipids of AP and EM were determined by HPTLC, and the fatty acid profile from AP, EM and plasma using TLC-FID. The relative amount of the lipid species analyzed in AP was in line with the data in the literature [phospholipids: 23.5 mol% more or less 3.5; total cholesterol 68.9 mol% more or less 7.9; triglyceride 7.6 mol% more or less 3.4]. Plasma and EM from CHD patients compared to controls, showed a decrease in polyunsaturated fatty acids and an increase in saturated fatty acids leading to a decrease in the unsaturation index (plasma: 1.67 more or less 0.06 vs. 1.28 more or less 0.03, P less than 0.05; EM: 2.28 more or less 0.04 vs. 1.25 more or less 0.010, P less than 0.05) and an enhancement in the saturated/unsaturated ratio (plasma: 0.35 more or less 0.02 vs. 0.52 more or less 0.02, P less than 0.05; EM: 0.45 more or less 0.01 vs. 0.83 more or less 0.04, P less than 0.05). These data are consistent with an essential fatty acid deficiency. Total cholesterol was increased in the CHD's EM (32.3 more or less 0.8 vs. 40.6 more or less 2.5, P less than 0.05) with a decrease in phospholipid percentage (67.7 more or less 0.7 vs. 59.4 more or less 2.6, P less than 0.05) indicating an alteration in membrane fluidity. These findings suggest changes in EM lipids in CHD patients in spite of different pathological conditions such as age, smoking status and diabetes. The analysis of the lipid composition of EM could provide a useful tool to monitor the evolution of the CHD.

El objetivo fue analizar la composición lipídica de las membranas de eritrocitos (ME), plasma y placas ateromatosas (PA) en pacientes con enfermedad coronaria avanzada (ECV). Las PA fueron obtenidas de endarterectomías coronarias de 18 pacientes. Fueron seleccionados 10 sujetos sanos, normolipémicos, como grupo control. Los lípidos totales de PA y ME se determinaron utilizando HPTLC, y el perfil de ácidos grasos de las PA, ME y plasma mediante TLC-FID. La cantidad relativa de las especies lipídicas obtenidas de las PA coinciden con la literatura [fosfolípidos 23.5 mol% más o menos 3.5; colesterol total 68.9 mol% más o menos 7.9; triglicéridos 7.6 mol% más o menos 3.4]. En el plasma y en las ME de los pacientes con ECV se observó, comparando con los pacientes controles, una disminución de los ácidos grasos poli-no saturados acompañado de un aumento de los ácidos grasos saturados que provocó el descenso del índice de instauración (plasma: 1.67 más o menos 0.06 vs. 1.28 más o menos 0.03, P menor que 0.05; ME: 2.28 más o menos 0.04 vs. 1.25 más o menos 0.010, P menor que 0.05) y el incremento del cociente AG saturados/insaturados (plasma: 0.35 más o menso 0.02 vs. 0.52 más o menos 0.02, P menor que 0.05; ME: 0.45 más o menos 0.01 vs. 0.83 más o menos 0.04, P menor que 0.05). Estos datos indicarían una deficiencia de ácidos grasos esenciales. Se observó una elevación en los valores de colesterol total (32.3 más o menos 0.8 vs. 40.6 más o menos 2.5, P menor que 0.05) y una disminución de los valores de fosfolípidos (67.7 más o menos 0.7 vs. 59.4 más o menos 2.6, P menor que 0.05) en las ME de los pacientes con ECV. Estos hallazgos sugieren cambios en los lípidos de las ME en los pacientes con ECV a pesar de presentar diferencias con respecto a edad, tabaquismo y diabetes. El conocimiento del perfil lipídico de las ME podría constituirse en una herramienta útil para monitorear la evolución de la enfermedad.

Trasplante hepático ortotápico en ratas. Técnica quirúrgica, complicaciones y sus tratamientos / Orthotopic liver transplantation in rats

The orthotopic rat liver transplant model is a widely used technique in transplantation research. It has many advantages over other animal transplant models because of its availability and low cost. However, it must be emphasized that success with the rat model requires thorough training. The aim of this paper is to describe the microsurgical technique involved in 60 rat liver transplants and to discuss the complications and their treatments. Forty-nine liver transplants were performed at the Experimental Laboratory of the University Hospital, Ontario, Canada (ELUH) and 11 were performed at the Laboratorio de Trasplante de Organos de la Facultad de Ciencias Medicas de La Plata, Buenos Aires. Argentina (LTO). Among the transplants performed at the ELUH, the observed complications were haemorrhage (n = 4), pneumothorax (n = 1), anastomotic failure (n = 15), bile leak (n = 3), and bile duct necrosis (n = 9). The remaining 17 rats at the ELUH were healthy at day 7 after surgery. Animal survival immediately postop, at 24 hours postop and at 7 days postop was achieved with the 9th, 20th and 21st transplants respectively. At the LTO, 3 rats died as a result of anaesthetic complications. Seven-day animal survival was achieved with the 11th transplant. We beleive that the description of the orthotopic rat liver transplantation technique, as well as the discussion regarding complications and their management, can be useful for researchers interested in performing liver transplantation in rats

[Orthotopic liver transplant in rats. Surgical technique, complications and treatment]. / Trasplante hepàtico ortotòpico en ratas. Técnica quirúrgica, complicaciones y sus tratamientos.

The orthotopic rat liver transplant model is a widely used technique in transplantation research. It has many advantages over other animal transplant models because of its availability and low cost. However, it must be emphasized that success with the rat model requires thorough training. The aim of this paper is to describe the microsurgical technique involved in 60 rat liver transplants and to discuss the complications and their treatments. Forty-nine liver transplants were performed at the Experimental Laboratory of the University Hospital, Ontario, Canada (ELUH) and 11 were performed at the Laboratorio de Trasplante de Organos de la Facultad de Ciencias Médicas de La Plata, Buenos Aires. Argentina (LTO). Among the transplants performed at the ELUH, the observed complications were haemorrhage (n = 4), pneumothorax (n = 1), anastomotic failure (n = 15), bile leak (n = 3), and bile duct necrosis (n = 9). The remaining 17 rats at the ELUH were healthy at day 7 after surgery. Animal survival immediately postop, at 24 hours postop and at 7 days postop was achieved with the 9th, 20th and 21st transplants respectively. At the LTO, 3 rats died as a result of anaesthetic complications. Seven-day animal survival was achieved with the 11th transplant. We beleive that the description of the orthotopic rat liver transplantation technique, as well as the discussion regarding complications and their management, can be useful for researchers interested in performing liver transplantation in rats.